Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

MICR222 > MODULE 5 - Putting Microbes to Work in the Environment > Flashcards

MODULE 5 - Putting Microbes to Work in the Environment Flashcards

1
Q

why do biofilms have to be on wet surfaces?

A

they have to have a moisture content so the bacteria can survive

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

what are some nutrients and environmental factors that form gradients?

A

water

pH

temp.

oxygen

pressure

radiation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

how can a bacteria survive starvation through morphological changes?

A

endospores (metabolically dormant, resistant to heat)

nucleoid condensation (nucleoid associated proteins bend DNA so it can condense, stress the bacteria and they switch to starvation mode where gene transcription changed, so stress response pathways activated and growth slowed)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

how can bacteria survive starvation with starvation proteins?

A

transpeptidases which cause peptidoglycan cross-linking and thickening of cell wall

chaperones which prevent denaturation and help renature damaged proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

why are starved cells hard to kill?

A

the can survive for years

can become more virulent

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

how does formation of persister cells occur?

A

small subset of cells which are spontaneously dormant (non-growing) even with nutrients available

starvation triggers persister cells through nutrient depletion leading to stress adaptation

formation of persister cell occurs in exponential phase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

why are persister cells important?

A

they can’t be treated with antibiotics

they don’t harbour antibiotic ressitance genes and instead express antibiotic tolerance phenotype

they are tolerant to the immune system and so are all together hard to eradicate

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

what is the difference between antibiotic resistance and antibiotic tolerance?

A

resistance based on genetic mutation on chromosome, while tolerance means the bacteria overcomes the antibiotic but isn’t resistant to it

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

what’s worse than having persister cells infecting you?

A

having persister cells in a biofilm infecting you

biofilm formation can also enhance persister cell formation by stressing bacteria

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

what are the stages of biofilm development?

A

reversible attachment

irreversible attachment (EPS starts to be secreted)

maturation

maturation and dispersion

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

where in the biofilm are you likely to find bacteria with a tolerant phenotype?

A

in the deeper levels where they are under more stress (O2 and nutrient limitations)

when bacteria don’t grow that well they might randomly start mutating chromosome, if antibiotic treatment happening they especially can develop R genes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

can biofilms have multiple type of microorganism in them?

A

yes

they can be individual or polymicrobial (e.g. bacteria, fungi)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

in what stage of biofilm formation are bacteria most vulnerable to eradication?

A

during reversible attachment

during irreversible attachment bacteria much more tolerant to antibiotics, immune system and starvation and generally cannot be eradicated by antibiotics alone

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

what is extracellular polymeric substances and what does it do?

A

major component of a biofilm (50-95% of dry weight)

chemical composition may vary between different strains/enviros but is primarily polysaccharide

protects from desiccation, antibiotics, toxins, immune cells

maintains integrity of biofilm and binds essential nutrients (making local rich environment)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

what are the advantages of biofilms?

A

physical attachment (in moving enviros like river, GI tract, bloodstream)

this beneficial as allows it to stay in one place, substrate may provide nutrients, extracellular enzymes that solubilise don’t get diluted quickly, nutrients may be higher in biofilm than enviro

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

what is the medical significance of biofilms?

A

delay wound healing

increase risk of infection (chronic, polymicrobial)

protects from body’s immune response (inflam response may induce biofilm formation)

providing nutrients in form of exudate (from dead immune cells)

damages healing tissue

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

how common are biofilm bacteria involved in chronic (non-healing) wounds?

A

biofilm bacteria in 60-90% chronic wounds

these wounds are stuck in inflammatory phase of healing and cannot progress further

often not easily realised and bacteria feed on host response

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

how can host immune response worse biofilm formation in wound?

A

neutrophils and macrophages come to wound to release proteases (e.g. matrix metalloproteinases (MMPs)) as these degrade dead tissue and extracellular matrix proteins

problem is when too many pathogens in wound more and more neutrophils come and produce more metalloproteases which provides more nutrients for bacteria by degrading dead tissue

in normal physiological conditions MMP levels controlled

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

what are matrix metalloproteinases (MMPs)?

A

proteases which require metal as co-factor for catalytic activity to occur

factor in abnormal healing in chronic wounds due to over production and increased protease activity

unbalanced activity facilitates colonisation and proliferation in chronic wounds

bacteria also secrete them so in bad wound lots of tissue being degraded thus lots of nutrients for bacteria

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

how are biofilms a problem for our teeth?

A

dental plaque is a biofilm and formation damages tooth and causes receding gums and bad breath

causes periodontal disease, dental caries

biofilms on teeth a symptom

flossing/brushing regularly prevents this

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

what are some biofilm infections that form from objects?

A

catheter insertion causing UTIs, vascular disease (bloodstream)

catheter contaminated often through caregivers hand

piercings, tattoos and brandings

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

how can we prevent biofilm formation on wounds?

A

regularly clean and debride wound tissue, remove biofilm material, necrotic tissue, foreign material in order to prevent biofilm maturation

sometimes difficult as biofilms might be attached to healthy tissue

most effective in early stages

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

how can we manage biofilm once it has formed in a wound?

A

increase frequency of debridement

cannot completely remove biofilm

once removed prevent re-establishment, otherwise this can happen within 24h

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

how do we treat bacterial biofilms once they are formed in wounds?

A

biofilms become resistant to most antimicrobials within 48-96hrs

clinical management requires complete removal of infected area

if not possible, attacking on regular schedule may force biofilm detachment, make bacteria susceptible for treatment and host defences HOWEVER can cause systemic infection e.g. sepsis, bacteraemia which are more virulent infections

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

what are the stages of biofilm development?

A

initial attachment phase

primary colonisation phase

climax community phase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

what influences adhesion of the first few bacteria in a biofilm?

A

linked to environmental cues and quorum sensing

they also produce biosurfactants to break down water content in an environment

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

what are the surfaces bacteria adhere to before forming biofilm?

A

attachment often occurs on rough surfaces e.g. a crack to get stuck in

if on smooth surface could be due to flow boundary layer where flow pushes bacteria against surfaces

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

describe the initial attachment phase?

A

microbes attach to a surface or themselves

attachment helped with flagella, filaments (help bacteria recognise surface structures), fimbriae (little charged hairs which are attracted to certain surfaces) and pili

mobile bacteria in aqueous phase become loosely attached via electrostatic or hydrophobic interaction w surface

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

describe the primary colonisation phase?

A

permanent attachment and symbiotic community

production of EPS and extracellular DNA which is the foundation of the biofilm

interaction with substrate which is mediated by production of extracellular polymers by colonising bacteria

once attached to surface active growth begins, micrcolonies form on substrate, further growth continues to cover entire surface

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

describe the climax community phase of biofilm development?

A

develops within seven days, growth continues until steady state

once mature biofilm may contain bacteria that can’t even attach to substrate or survive initial nutritional restrictions but other bacteria clutched up (polymicrobial)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

what is biofouling?

A

accumulation (usually a biofilm) of bacteria on a surface they are not wanted on e.g. industrial equipment, ship

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

what is economies of scale?

A

ideally you spend lots on equipment at first and then over time produce more product and so it pays off the investment

so you have to find sweet spot where investment pays off as equipment lasts longer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

what are common places in industry where biofilm formation is a problem?

A

power plants, air conditioning, food processing, oil refining

biofilms cause major production disruptions

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
34
Q

what are some industrial examples of financial losses due to biofilms?

A

food industries lose large production due to contamination

fouling of pipes leads to friction and plant failure through corrosion and lesser flow

heat exchanger surfaces lead to inefficient heat exchange

cleaning requires closing of entire plant and scrubbing of plumbing which decreases production

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
35
Q

how are biofilms a major problem for the dairy industry?

A

milk highly perishable as has high nutrient content so easy for microbes to grow

sterile in udder cells

biofilms form at air-liquid interface and are usually bacillus species (floating biofilms)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
36
Q

outline the bacillus genus and how it is bad for dairy industry?

A

gram-positive rod shaped

highly detrimental as cause dairy spoilage and illness

associated with animal udders and then spread through diary production systems

prod heat resistant endospores allowing persistence through pasteurisation and then biofilm formation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
37
Q

what are some biofilm components produced by bacillus?

A

exopolysaccharides

amyloid-like fibres (non-soluble proteins) - soluble proteins which fold into insoluble fibres allowing further resistance

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
38
Q

what are some common methods of cleaning equipment for biofilm control and what are some problems with this?

A

use cheap chemical agents such as chlorine, NaOH, acid

chlorination is most common methods however problem is some microbes resistant and chlorine also not good enough to eliminate biofilms as leaves EPS matrix intact

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
39
Q

what are some examples of damage caused to at home equipment from biofilms?

A

rubber seals in washing machine and dishwasher

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
40
Q

what is the dairy plant cleaning in place (CIP) procedure?

A

cold water and acid wash immediately after milking

hot water and NaOH wash to remove adhered residues at least twice a week

cold water acid wash after each alkali wash to remove minerals, kill remaining bacteria and neutralise alkali solution

final wash w detergent

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
41
Q

how acids destroy cells?

A

denature proteins, extremely reactive and break apart chemical structures, upon exposure cells die but don’t disintegrate

42
Q

how do alkalis destroy cells?

A

accept protons (i.e. rip them the fuck off other molecules), found in cleaners and bleach

burns from alkalis way worse than acid burns as they liquify biological material

43
Q

how does biofouling cause problems in NZ involving marine pests and disease?

A

underside of bots prone to bacteria attachment and biofilm formation

this can spread harmful bacteria

to prevent this must clean hull of ship, also can use antifouling coating

vessels must show evidence of biofouling management

44
Q

outline some key forms of biofouling management?

A

clean niche areas e.g. protrusions, recesses in deck, unpainted parts of hull

maintenance procedures e.g. paint w antifouling paints, marine growth prevention systems (e.g. approved chemicals), steam blow-out pipes

45
Q

how are certain surfaces anti-fouling?

A

reagents/dispersants such as repelling agents, slippery surface coatings and nanoscale surface topologies

46
Q

how can biocides prevent fouling on the hulls of boats?

A

chemical removal of biofouling and can be incorporates into antifouling coatings

specifically kills bacteria, fungi and algae

e.g. tributyltin (TBT)

47
Q

what is tributyltin (TBT)?

A

a common biocide which is now banned

component of paint and slowly released into environment

very poisonous to marine life e.g. shellfish

highest concentrations in harbours where boats with this coating moored

48
Q

do antifouling coatings kill microorganisms?

A

nah they just prevent them from attachment

49
Q

what are the three main classes of antifouling coatings?

A

hydrophobic coating

hydrophilic coating

polymer coating

50
Q

what are hydrophobic coatings?

A

repel water

low friction and surface energy

smooth surface

problem is no mechanical strength and only short term stability (so have to be reapplied frequently)

e.g. silicone or umbrellas/rain jackets

51
Q

what are hydrophilic coatings?

A

attract water

highly hydrated zwitterions composed of glycine betaine get saturated w water molecules and repel organisms, enzymes and EPS

low friction and work better then hydrophobic surfaces in regards to biofilm formation

however not commercially available

52
Q

what are polymer coatings?

A

poly(ethylene glycol) - PEG

hydrophilic but water soluble so difficult to use as a coating in aqueous enviro

research on mussel adhesive proteins (MAPs) prod by blue mussel and adding this to PEG to make it more adhesive

53
Q

what are some antifouling examples from nature?

A

bacteria don’t attach to shark skin, butterfly wings, seaweed

so there are antimicrobial antifouling compounds in nature we haven’t discovered which could be mimicked for engineering purposes

54
Q

what are some coatings we could add to our teeth to prevent oral biofilms?

A

Microparticles w quorum sensing inhibitors to block biofilm formation

calcium-binding polymer coated microparticles

could allow sustained release of QS inhibitors

55
Q

how did oil get dispersed far as fuck when deepwater horizon happened?

A

grease-cutting soaps such as dispersants break oil into small bits and so it merged with deep sea water and sunk

it also spread as dragged down by marine snow and wind/currents pushed it far away

56
Q

when they couldn’t find like a quarter of the oil spilt from deepwater horizon, what happened to it?

A

hydrocarbon-degrading bacteria in the sediments mineralised it to CO2 or immobilised it as biomass

57
Q

what is bioremediation?

A

engineered process using microbes to break down environmental pollutants from a spillage

microbes involved are often extremophiles as they can tolerate high levels of organic solvents

58
Q

what are hydrocarbonoclastic bacteria?

A

two main species: Alcanivorax borkumensis
Oleispira antarctica

can break down hydrocarbons to CO2 and energy however cannot break down polyaromatic hydrocarbons as complex structure

require specific O2, nitrogen, phosphorus and temp to live and often this not correct in oil spill zones of ocean

59
Q

describe Alcanivorax borkumensis?

A

aerobic hydrcarbonoclastic bacterium which propagates in seawater containing crude oil

oil leakage increases phosphorous and nitrogen which is natural nutrient for bacteria = better growth

produces biosurfactants which break down water surface tension so oil forms droplet

bacterial then covers droplets w biofilm

60
Q

describe Oleispira antarctica?

A

aerobic/anaerobic hydrocarbonoclastic bacterium which lives in cold marine water

can degrade oil in cold and deep water

growth temp. between 1-15 degrees celsius

61
Q

what are some examples of fungi we could use for bioremediation?

A

oyster mushroom - eats diesel and other petroleum products producing CO2 and H2O as result

white rot fungus - can break down polyaromatic hydrocarbons that the bacteria couldnt prod H2O2

62
Q

what are some of the drawbacks of using fungi for bioremediation?

A

they grow slow af so slow process

they will also degrade biological material so other environmental consequences (this one applies to bacteria too)

63
Q

what are the two types of bioremediation?

A

biostimulation - environmental modification by adding nutrients to stimulate certain microbes or aerating soil/water

bioaugmentation - addition of microbes

64
Q

what are the advantages of biostimulation?

A

cheaper

natural process so publicly acceptable

less disturbing to enviro

destroys wide range of contaminants

65
Q

what are the disadvantages of biostimulation?

A

often unpredictable or unsuccessful

physical environment challenging due to pH, temp, O2 etc. (so adding nutrients to certain enviro may not help growth as other factors limiting)

concentration dependent

slow process

biological competition

66
Q

what are some problems with bioaugmentation?

A

bacteria require certain nutrients that might not be present

not enough bacterial numbers and they just get diluted in enviro

could disturb ecological niches

67
Q

what is bioaugmentation good for?

A

degrading specific soil or groundwater contaminants such as chlorinated solvents or petroleum

68
Q

describe the Pseudomonas species?

A

rod-shaped bacterial species

opportunistic pathogen

some can protect crops from pests and disease

some can cause precipitation by cooling vapour and some can utilise petroleum products

so can be used to clean up spills

69
Q

what is biodegradation?

A

natural break down of everything which replenishes ecosystem

microbes degrade organic matter slowly

70
Q

what are the stages of biodegradation?

A
  1. biodeteriation - surface level degradation where material properties modified
  2. biofragmentation - degrades polymers to oligomers and monomers; if anaerobic prod methane, if aerobic prod CO2 and H2O
  3. assimilation/mineralisation - microbes use the products of prev step
71
Q

what are some common plastic degrading microbes?

A

Penicillum, Aspergillus, Pseudomonas sp.

these can degrade non-biodegradable plastics and consume for growth by secreting PETase enzymes

slow degradation time so no commercial scale

also some gut microbes and also mealworms

72
Q

what are xenobiotics?

A

foreign compounds introduced into environment by humans

73
Q

give an example of a xenobiotic and how it is problematic?

A

DDT is a pesticide which kills insects and thus can control malaria and typhus outbreaks

still present in soil, has detrimental reproductive impacts (oestrogen blocker) and causes cancer

74
Q

why are xenobiotics usually so hard to get rid of?

A

they usually stable for a long time

have complex ring structures and stable C-halogen bonds which are highly chlorinated

very recalcitrant to microbial attack

shortage of enzymes to get rid of them

75
Q

how can we degrade DDT?

A

reduce the organochlorines (used in pesticides)

DDT converted to DDE and then to DDD

this can be done with sulcate-reducing bacterium such as Clostridium sp.

76
Q

what is PCB?

A

a very chlorinated and very recalcitrant xenobiotic used in coolant fluids

it may not degrade at all

takes a long time to test for biodegradation

77
Q

what happens to waste material from an organism in the environment?

A

either used directly by another organism or converted

78
Q

what are common liquid wastes humans produce?

A

shower water

urine

washing-machine water

trade wastes and other industrial wastes

79
Q

what are some solid wastes humans produce?

A

human faeces

kitchen waste

(nowhere near as much liquid waste and gets mixed w water for transport e.g. flush toilet)

80
Q

what is mixed domestic sewage?

A

our combined liquid and solid wastes

81
Q

what does waste water include and what are there in high levels?

A

includes sewage, industrial and agricultural effluent, street runoff collected in drains

contains high levels of organic matter, heavy metals, nutrients and microorganisms

82
Q

what are some common diseases that are spread in wastewater?

A

typhoid

cholera

COVID-19 (not really common but relevant)

83
Q

what are the main microbes of human origin in wastewater?

A

coliforms such as E. coli

presence of E. coli indicates faecal matter from warm-blooded animals (hence use as indicator bacteria)

some disease causing pathogens e.g. Salmonella can spread and re-enter human food chain e.g. through shellfish

can contaminate groundwater

84
Q

what are the goals of wastewater treatment?

A

eradicate any human pathogen

seperate wastewater into sludge and a dissolved fraction containing water, organic material, bacteria and salts

reduce organic load (microbes, insoluble debri, soluble organic matter)

remove chemical compounds e.g. N and P

make it safe for marine disposal

85
Q

what occurs in primary treatment of wastewater?

A

wastewater enters treatment plant

passed through milliscreens which sieve for large solids (anything that doesn’t go through goes to landfill)

then through grit chambers which slow the flow and into primary clarifier

86
Q

what does the primary clarifier do?

A

separates suspended solids from liquid

gravity pulls smaller particles into sludge and lighter solids float at top and accumulate as scum

sludge goes into waste disposal area

87
Q

what occurs in secondary treatment of wastewater?

A

aeration basins blow air in to keep mixture in motion and provides oxygen

environmentally friendly bacteria (large consortia) feed on nutrients in wastewater e.g. fats, sugar. ammonia

bacterial clusters form as they break down waste; can form biofilms which are removed via settlement

secondary clarifier separates bacterial clusters from liquid through flocculation

activated sludge pumped back into aeration basin to provide more bacteria for breakdown

separation of solids from liquids and then centrifugation (liquid back into aeration basin and sludge disposed)

88
Q

what occurs in final/tertiary treatment?

A

disinfection to kill any remaining microorganisms; traditionally used chlorine (effluent <0.5mg/L Cl)

filtration (e.g. gravel or sand)

UV light disinfection

release into harbour

89
Q

how can we find microbes for remediation?

A

isolation and characterisation of microbes from unique habitats

traditional lab conditions that test microbial consortia to perform bioremediation

culture-based techniques (problematic cause so many microbes unculturable)

DNA-based techniques are what we now turning towards

90
Q

what are some DNA-based techniques for identification of microorganisms?

A

16S rRNA sequencing

Omics-approaches

91
Q

what are the omics-approaches?

A

discover novel microbes not accessible w traditional culturing

explore metagenomes of contaminated environmental samples for their microbial communities (gives insight on diversity)

allows to design and develop efficient strains of microbes e.g. for better metabolism of xenobiotics

92
Q

what is Pseudomonas putida?

A

soil bacterium and plant coloniser

adapted lifestyle to harsh environmental conditions and stresses

remarkable metabolic and physiological robustness

uses wide variety of C and N sources

can use omics to engineer new optimised strains

93
Q

what is metabolic engineering?

A

purposeful modification of cellular networks including metabolic, gene regulatory, and signalling networks to achieve desirable goals e.g. enhanced production of metabolites, increase intracellular levels of essential precursors, nutrient source uptake and by-product formation

94
Q

how can metabolically engineer Pseudomonas putida for better bioremediation?

A

synthetic biology tools combined with omics data can be used to re-purpose the central carbon metabolism leading to synthesis of new chemical structures

95
Q

what are some problems with bioengineering and how can we overcome them?

A

production of dead-end substrates e.g. reactive intermediates which cannot be metabolised so bacteria stop growing

overcome by restructuring existing pathways and developing new ones with different enzymes

95
Q

what are some problems with bioengineering and how can we overcome them?

A

production of dead-end substrates e.g. reactive intermediates which cannot be metabolised so bacteria stop growing

overcome by restructuring existing pathways and developing new ones with different enzymes

96
Q

what are DCA and TCP examples of?

A

carcinogenic and mutagenic xenobiotics which cause reproductive effects

highly recalcitrant in enviro and initially used as degreasing agent

spread via groundwater flows

97
Q

how can bacterial degradation of DCA occur and how could we use this for large scale industrial applications?

A

Xanthobacteri autotrophicus pathway can degrade it by:

haloalkane dehalogenase hydrolyses one C-Cl bond

dehydrogenase produces chloroacetic acid

dehalogenase converts this to glycolic acid which then can turn to CO2

if we introduce this pathway into P. putida could be used for large scale applications

98
Q

how can bacterial degradation of TCP occur?

A

natural organisms cannot mineralise TCP, however similarly structured compounds are biodegradable

protein and metabolic engineering to construct microbes with improved catabolic activities

99
Q

how could we engineer DCP degrading enzymes to degrade TCP?

A

haloalkane dehalogenase (DhIA) catalyses first step in DCA degradation and could be engineered to have wider substrate range

this engineering could be done through site-directed mutagenesis to improve activity or modification of tunnel proteins

100
Q
A

very strict here but some techniques no approved e.g. more protein-protein expression which is way more safe and cost effective than other methods

MICR222 (6 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions